Polyanionic non-collagenous proteins and their analogues promote artificial mineralization of embryonic mouse bone

Non-collagenous proteins (NCPs) are specialized biomacromolecules within the extracellular matrix (ECM) that regulate the mineralization of calcified tissues, such as bone and dentin. Numerous in vitro studies have demonstrated that natural polyanionic NCPs and their analogues can mediate intrafibrillar mineralization, characterized by the infiltration of apatite minerals into collagen fibrils. However, these studies primarily utilize self-assembled collagen fibrils or demineralized mature tissues, leaving it unclear whether pristine embryonic bone ECM at a developmental stage permissive to mineral deposition can regulate intrafibrillar mineralization independently or requires polyanionic NCP substitutes to promote the process artificially. To address this, we employed an ex vivo model of endochondral ossification using metatarsals isolated from 15-day-old embryonic mice (E15). In addition to a supersaturated calcium (Ca) and inorganic phosphate (Pi) medium, we introduced fetuin-A, a native polyanionic NCP or poly-DL-aspartic acid (pAsp), commonly used as an NCP substitute. The incorporation of either additive was essential for the effective mineralization of embryonic metatarsals. Both fetuin-A and pAsp played a direct role in facilitating the infiltration of Ca-Pi precursors into the avascular cartilaginous matrix. Raman spectroscopy and electron microscopy confirmed the formation of hydroxyapatite (HAp) exhibiting diverse levels of crystallinity, with fetuin-A supplementation resulting in the greatest HAp accumulation within the rudiments. HAp was localized in the perichondrium, a region conducive to initial mineralization and enriched with a fibrillar network of collagen types I and II. Three-dimensional reconstructions implementing Dijkstra’s algorithm revealed the association between HAp and collagen fibrils either organized in an intrafibrillar, extrafibrillar, or combined arrangement.